Enkephalin Analogue Research Articles

Delta Opioid Peptide Augments Functional Effects and Intrastriatal Graft Survival of Rat Fetal Ventral Mesencephalic Cells

Delta enkephalin analogue [D-Ala(2),D-Leu(5)]enkephalin (DADLE) has been shown to protect dopamine transporters from methamphetamine-induced neurotoxicity. In the present study, we demonstrate that exposure of embryonic ventral mesencephalic cells to DADLE (0.01 g/ml), prior to intrastriatal transplantation, enhanced functional recovery and graft survival in 6-hydroxydopamine-induced hemiparkinsonian rats. At 6 and 8 weeks posttransplantation, animals that received DADLE-treated cell grafts exhibited significantly higher (near normal) spontaneous locomotor behaviors, as well as trends of greater reversal of motor asymmetrical behaviors compared with animals that received nontreated cell grafts. Histological examination revealed that animals transplanted with DADLE-treated cell grafts exhibited about twice the number of surviving tyrosine hydroxylase-immunoreactive grafted neurons compared with those animals that received nontreated cell grafts. These results suggest that DADLE should be considered as an adjunctive agent for neural transplantation therapy in Parkinson's disease.

Synthesis and evaluation of N,N-dialkyl enkephalin-based affinity labels for delta opioid receptors.

To develop affinity labels for delta opioid receptors based on peptide antagonists, the Phe(4) residues of N,N-dibenzylleucine enkephalin and N,N-diallyl[Aib(2),Aib(3)]leucine enkephalin (ICI-174, 864) were substituted with either Phe(p-NCS) or Phe(p-NHCOCH(2)Br). A general synthetic method was developed for the conversion of small peptide substrates into potential affinity labels. The target peptides were synthesized using Phe(p-NH(2)) and a Boc/Fmoc orthogonal protection strategy which allowed for late functional group conversion of a p-amine group in the peptides to the desired affinity labeling moieties. A key step in the synthesis was the selective deprotection of a Boc group in the presence of a tert-butyl ester using trimethylsilyl trifluoromethanesulfonate (TMS-OTf). The target peptides were evaluated in radioligand binding experiments in Chinese hamster ovary (CHO) cells expressing delta or mu opioid receptors. The delta receptor affinities of the N, N-dibenzylleucine enkephalin analogues were 2.5-10-fold higher than those for the corresponding ICI-174,864 analogues. In general, substitution at the para position of Phe(4) decreased binding affinity at both delta and mu receptors in standard radioligand binding assays; the one exception was N, N-dibenzyl[Phe(p-NCS)(4)]leucine enkephalin (2) which exhibited a 2-fold increase in affinity for delta receptors (IC(50) = 34.9 nM) compared to N,N-dibenzylleucine enkephalin (IC(50) = 78.2 nM). The decreases in mu receptor affinities were greater than in delta receptor affinities so that all of the analogues tested exhibited significantly greater delta receptor selectivity than the unsubstituted parent peptides. Of the target peptides tested, only N, N-dibenzyl[Phe(p-NCS)(4)]leucine enkephalin (2) exhibited wash-resistant inhibition of radioligand binding to delta receptors. To our knowledge, 2 represents the first peptide-based affinity label to utilize an isothiocyanate group as the electrophilic affinity labeling moiety. As a result of this study, enkephalin analogue 2 emerges as a potential affinity label useful for the further study of delta opioid receptors.

Spectroscopic evidence of β-turn in N-glycated peptidomimetics related to leucine–enkephalin

The conformational differences caused by N-glycation of the amide bond in endogenous opioid pentapeptide leucine–enkephalin (Tyr–Gly–Gly–Phe–Leu) have been explored in solution using FTIR spectroscopy, NMR and molecular modelling. The compounds studied include protected and unprotected enkephalin analogues N-alkylated at the second (Gly 2) amino acid residue with a 6-deoxy- d-galactose moiety ( 1– 3). Comparison of the amide I component bands in the FTIR spectra, measured in trifluoroethanol (TFE), CHCl 3 and DMSO, revealed significant differences in the intensity as well as shifts in component band frequencies for glycopeptides 1– 3. We found that only the FTIR spectrum of the fully protected compound 1 indicated the presence of a higher population of β-turns, while the spectra of the partially protected and unprotected glycopeptides 2 and 3 reflected the dominance of unordered or open structures, with some low population of turns. The observed NOE connectivities in CDCl 3 for both isomers of the fully protected compound 1, the all-trans one and another with Tyr 1–Gly 2 peptide bond in cis conformation, indicate the presence of a β-like turn conformation. Molecular dynamics simulations of the glycopeptide 1 obtained by unconstrained energy minimization of trans- and cis- 1 shows that one of trans form conformations is consistent with β-turn whereas cis isomer has revealed less-compact turn.

Optically active aromatic amino acids. Part VI. Synthesis and properties of (Leu5)-enkephalin analogues containing O-methyl-L-tyrosine1 with ring substitution at position 3'.

Twelve new [Tyr(Me)1, Leu5]-enkephalin analogues with substituents at position 3' of the Tyr ring have been synthesized using traditional solution methods. The substituents were -CO2H, -CONH2, -CO2Me, -(E)-CH=NOH, -(E)-CH=NOMe and CH2OH. The analogues were C-terminated with methyl esters, amides or as free acids. In the in vitro biological assays a remarkable agonist activity to the opiate receptor mu in guinea pig ileum (GPI) relative to Leu-ENK was shown by the following: Leu-ENK, 100; [Tyr(Me)(3'-CO2Me)1, Leu-OMe5]-ENK (I), 8.1; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OMe5]-ENK (VI), 26.2; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OH5]-ENK (VII), 2.9; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-NH2(5)]-ENK (VIII), 4.7; and [Tyr(Me)(3'-CH2OH)1, Leu-OMe5]-ENK (X), 5.6. The agonist effect was naltrexone- or naloxone-reversible. The masking of the hydroxyl group in (E)-hydroxyiminomethyl group of analogue (VI) by O-methylation has totally abolished its GPI agonist activity. It seems that the (E)-CH=NOH group shows affinity and plays an analogous role to the phenol group Tyr1 in leucine-enkephalin and in the tyramine group of the opiate alkaloids. The analogues: [Tyr(Me)(3'-CO2Me)1, Leu-OMe5]-ENK (I), [Tyr(Me)(3'-CO2H)1, Leu-OMe5]-ENK (II), [Tyr(Me)(3'-CO2Me)1, Leu-NH2(5)]-ENK (III), [Tyr(Me)(3'-CO2H)1, Leu-NH2(5)]-ENK (IV), [Tyr(Me)(3'-CONH2)1, Leu-NH2(5)]-ENK (V), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OMe5]-ENK (VI), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OH5]-ENK (VII), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-NH2(5)]-ENK (VIII), [Tyr(Me)(3'-(E)-CH=NOMe)1, Leu-OMe5]-ENK (IX), [Tyr(Me)(3'-CH2OH)1, Leu-OMe5]-ENK (X), [Tyr(Me)(3'-CH2OH)1, Leu-OH5]-ENK (XI) and [Tyr(Me)(3'-CH2OH)1, Leu-NH2(5)]-ENK (XII) under testing had no significant agonist activity to the enkephalinergic receptor in mouse vas deferens (MVD). All methyl esters of synthesized analogues of [Leu5]-ENK showed higher activity to mu receptors than structurally identical C-terminal amides. It is a surprising result since usually C-terminate amides are stronger agonists than C-terminate esters.

Treatment with delta opioid peptide enhances in vitro and in vivo survival of rat dopaminergic neurons.

A major problem in neural transplantation therapy is poor survival of grafted cells, which may be due to low cell viability prior to transplantation or scarce trophic factors available to the cells following transplantation. Recently, the delta enkephalin analogue [D-Ala(2),D-Leu(5)]-enkephalin (DADLE) has been demonstrated to protect against, as well as to reverse methamphetamine-induced loss of dopamine transporters. Here, we show that pretreatment with DADLE (0.0025, 0.005, 0.01 g/ml) dose-dependently enhanced cell viability of cultured primary rat fetal mesencephalic cells. In addition, DADLE administration in adult rats (4 mg/kg every 2 h, 4 injections, i.p.) prior to 6-hydroxydopamine lesions of the medial forebrain bundle, significantly reduced the severity of loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra 1 month post-lesion. This is the first report suggesting that DADLE can be used as a supplement factor for improving the cell viability of fetal mesencephalic cells and as a protective agent against neurotoxicity in a Parkinson's disease model.

Cyclopropane-derived peptidomimetics. Design, synthesis, and evaluation of novel enkephalin analogues.

It is known that peptide mimics containing trans-substituted cyclopropanes stabilize extended conformations of oligopeptides, and molecular modeling studies now suggest that the corresponding cis-cyclopropane dipeptide isosteres could stabilize a reverse turn. To begin to assess this possibility, a series of cis-substituted cyclopropanes were incorporated as replacements of the Gly(2)-Gly(3) and Phe(4)-Leu(5) dipeptide subunits in Leu-enkephalin (H(2)N-Tyr-Gly-Gly-Phe-Leu-OH), which is believed to bind to opiod receptors in a conformation containing a beta-turn. General methods for the synthesis of the cyclopropane-containing dipeptide isosteres -XaaPsi[COcpCO]Yaa- and -XaaPsi[NHcpNH]Yaa-were developed by a sequence that featured the enantioselective cyclization of allylic diazoacetates catalyzed by the chiral rhodium complexes Rh(2)[(5S)-MEPY](4) and Rh(2)[(5R)-MEPY](4). A useful modification of the Weinreb amidation procedure was applied to the opening of the intermediate lactones with dipeptides, and a novel method for the synthesis of substituted diaminocyclopropanes was also developed. The Leu-enkephalin analogues were tested in a panel of binding and functional assays, and although those derivatives containing cyclopropane replacements of the Gly(2)-Gly(3) exhibited low micromolar affinity for the mu-receptor, analogues containing such replacements for the Phe(4)-Leu(5) subunit did not bind with significant affinity to any of the opioid receptors. These results are discussed.

Development of novel lipophilic derivatives of DADLE (leucine enkephalin analogue): intestinal permeability characateristics of DADLE derivatives in rats.

The objective of this study is to examine the intestinal permeability of novel lipophilic derivatives of DADLE (Tyr-D-Ala-Gly-Phe-D-Leu), an enkephalin analogue, using isolated rat intestinal membranes. The novel lipophilic derivatives of DADLE were synthesized by chemical modification with various fatty acids at the C terminus. The pharmacological activities of these DADLE derivatives were assessed by a hot plate test. The intestinal permeability of these derivatives was estimated by the in vitro Ussing chamber method. We obtained four different DADLE derivatives including acetyl-DADLE (DADLE-C2), butyryl-DADLE (DADLE-C4), caproyl-DADLE (DADLE-C6), and caprylyl-DADLE (DADLE-C8). All the derivatives of DADLE had at least 75% of the activity of native DADLE, suggesting that chemical modification of DADLE at the C terminus did not markedly affect its pharmacological activity. These DADLE derivatives were more stable than native DADLE in jejunal and colonic homogenates. A "bell-shaped" profile was observed between the apparent permeability coefficients (Papp) of DADLE derivatives and lipophilicity. In particular, DADLE-C4 had the greatest permeability characteristics across the intestinal membrane of the acyl derivatives studied in this experiment. The permeability of DADLE-C4 across the jejunal membrane was further improved in the presence of puromycin, amastatin, and sodium glycocholate (NaGC), all at a concentration of 0.5 mM. We suggest that the combination of chemical modification with butyric acid and the application of a protease inhibitor are effective for improving the absorption of DADLE across the intestinal membrane.

Enkephalin-based drug design: conformational analysis of O-linked glycopeptides by NMR and molecular modeling

Glycosylation provides an effective means of enhancing penetration of the blood–brain barrier by pharmacologically active peptides. Glycosylated enkephalin analogues demonstrate much greater analgesic effects than their unglycosylated counterparts when administered peripherally. The solution conformations of glycopeptide enkephalin analogues with the sequences H-Tyr-c-[ d-Cys-Gly-Phe- d-Cys]-Ser(β- O-Glcp)-Gly-NH 2, 2, and H-Tyr-c-[ d-Cys-Gly-Phe- d-Cys]-Ser(α- O-Glcp)-Gly-NH 2, 3, have been determined by NMR and molecular modeling, and were compared to the unglycosylated peptide H-Tyr-c-[ d-Cys-Gly-Phe- d-Cys]-Ser-Gly-NH 2, 1, to determine the impact of glycosylation on peptide conformation. The only observed conformational effects were on the residue of attachment, Ser 6, and on the adjacent Gly 7-amide. This has important implications in peptide-based drug design in that strategically placed glycosylation can improve transport without destruction of the receptor selectivity of a pre-existing non-glycosylated peptide pharmacophore.

Enkephalin-cell interactions

Enkephalin analogs for multivalent ligand systems, bivalent enkephalins, multivalent enkephalins on polymers, multivalent ligands on vesicles, simultaneous activation of two different receptor systems, and cell interactions of enkephalin/polypeptide conjugates are described. Multivalent ligand systems can trigger receptor-receptor interactions and are considered to possess interesting possibilities in terms of enhanced potency, reduction of side effects, and a new biological activity.

Enkephalinergic Neurons in the Periaqueductal Gray and Morphine Withdrawal

The effects of opioid (e.g., morphine) withdrawal on levels of endogenous opioid peptides and their mRNA in the various brain regions have been studied. However, the role of this opioidergic mechanism in the mediation of opioid withdrawal is not fully understood. Preproenkephalin (PPE) mRNA in the caudal periaqueductal gray (cPAG), an important brain region in opioid withdrawal, is increased by both opioid antagonist (naloxone)-precipitated and spontaneous morphine withdrawal, but not by various other stresses in rats, indicating a role of endogenous enkephalins in the cPAG in morphine withdrawal. In addition, PPE mRNA levels in the cPAG increase in the course of the dissipation of morphine withdrawal, and they are returned to the control levels after disappearance of morphine withdrawal signs. Local administration of an enkephalin analog or peptidase inhibitors into the cPAG suppresses morphine withdrawal signs. These facts suggest that enkephalinergic neurons in the PAG may have a critical role in the recovery phase of morphine withdrawal. Recently, an involvement of transcription factors in morphine withdrawal has been suggested. Thus, the possible role of transcription factors in the regulation of PPE gene expression in the cPAG during morphine withdrawal is also discussed.

δ Opioid Receptor Modulation of Several Voltage-Dependent Ca2+Currents in Rat Sensory Neurons

Endogenous enkephalins and delta opiates affect sensory function and pain sensation by inhibiting synaptic transmission in sensory circuits via delta opioid receptors (DORs). DORs have long been suspected of mediating these effects by modulating voltage-dependent Ca(2+) entry in primary sensory neurons. However, not only has this hypothesis never been validated in these cells, but in fact several previous studies have only turned up negative results. By using whole-cell current recordings, we show that the delta enkephalin analog [D-Ala(2), D-Leu(5)]-enkephalin (DADLE) inhibits, via DORs, L-, N-, P-, and Q-high voltage-activated Ca(2+) channel currents in cultured rat dorsal root ganglion (DRG) neurons. The percentage of responding cells was remarkably high (75%) within a novel subpopulation of substance P-containing neurons compared with the other cells (18-35%). DADLE (1 microM) inhibited 32% of the total barium current through calcium channels (I(Ba)). A delta (naltrindole, 1 microM), but not a mu (beta-funaltrexamine, 5 microM), antagonist prevented the DADLE response, whereas a DOR-2 subtype (deltorphin-II, 100 nM), but not a DOR-1 (DPDPE, 1 microM), agonist mimicked the response. L-, N-, P-, and Q-type currents contributed, on average, 18, 48, 14, and 16% to the total I(Ba) and 19, 50, 26, and 20% to the DADLE-sensitive current, respectively. The drug-insensitive R-type current component was not affected by the agonist. This work represents the first demonstration that DORs modulate Ca(2+) entry in sensory neurons and suggests that delta opioids could affect diverse Ca(2+)-dependent processes linked to Ca(2+) influx through different high-voltage-activated channel types.

The effect of halogenation on blood–brain barrier permeability of a novel peptide drug☆

The utility of a drug depends on its ability to reach appropriate receptors at the target tissue and remain metabolically stable to produce the desired effect. To improve central nervous system entry of the opioid analgesic [ d-Pen 2, l-Pen 5, Phe 6]Enkephalin (DPLPE-Phe), our research group synthesized analogs that had chloro, bromo, fluoro, and iodo halogens on the para positions of the phenylalanine-4 residue. This study reports on investigation of the effect of halogenation on stability, lipophilicity, and in vitro blood–brain barrier permeability of a novel enkephalin analog DPLPE-Phe. The stability of each halogenated DPLPE-Phe analog as well as the amidated and nonamidated parent peptide was tested in plasma and brain. All peptides tested had a half-time disappearance >300 min except for DPLPE-Phe-NH 2, which was found to have a half-life of 30 min in plasma. Octanol/saline distribution studies indicated addition of halogens to DPLPE-Phe-OH significantly increased lipophilicity except for p-[F-Phe 4]DPLPE-Phe-OH. p-[Cl-Phe 4]DPLPE-Phe-OH exhibited the most pronounced increase in lipophilicity. Para-bromo and para-chloro halogen additions significantly enhanced in vitro blood–brain barrier permeability, providing evidence for improved delivery to the central nervous system.

Fluorescence decay time distribution analysis of cyclic enkephalin analogues. Influence of the solvents and configuration of amino acids in position 2 and 3 on changes in conformation.

The lifetime distribution calculations were applied to study the influence of configuration of amino-acid residues in positions 2 and 3 on changes in conformation of the peptide chain of cyclic analogues of enkephalins containing a fluorescence energy donor and acceptor in different solvents. In all the solvents studied the lifetime distributions were bimodal. This testified to the presence of two families of conformations. In this paper the relationship between the population of each conformation and configuration of the residues in position 2 and 3, and the solvent used is discussed.

Conformational analysis of a novel cyclic enkephalin analogue using NMR and EDMC calculations.

Conformational space of a novel cyclic enkephalin analogue, cyclo(N(epsilon),N(epsilon')-carbonyl-D-Lys2,Lys5)enkephalin amide, was exhaustively examined. A large number of conformations was selected and clustered into families on the basis of their structure and energy. For representative conformations ROESY spectra were generated and their linear combination was fitted to the spectra measured in water and Me2SO-d6. This procedure yielded an ensemble of most populated conformations of the peptide in the two solvents.

Circular dichroism study of the carbohydrate-modified opioid peptides

The conformational preferences of enkephalins and the related glycoconjugates in which free or protected carbohydrate moieties were linked to the opioid peptides through an ether, ester or amide bond were investigated by circular dichroism spectroscopy in water, trifluoroethanol and water-trifluoroethanol mixtures. The analysis of the spectra revealed that the conformation of the enkephalin molecule is very sensitive to slight changes in the peptide structure around the C-terminal region. It was found that the type II β-turn structures are populated in N-terminal tetrapeptide enkephalin fragment, while leucine-enkephalin amide feature a type I (III) β-turn structure in solution. Incorporation of the sugar moiety into opioid peptide compound did not significantly influence the overall conformation of the peptide backbone, although minor intensity changes may reflect shifts in the population of the different turn systems. These small structural alterations can be responsible for the receptor-subtype selectivity of the various carbohydrate-modified enkephalin analogs.

ChemInform Abstract: A New Efficient Method for S—CH2—S Bond Formation and Its Application to a Djenkolic Acid‐Containing Cyclic Enkephalin Analogue.

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Effect of the tetrazole cis-amide bond surrogate on the complexing ability of some enkephalin analogues toward Cu(II) ions

A study of the effect of the tetrazole moiety, a cis-amide bond surrogate, on the Cu(II) coordinating properties of oligopeptides is reported. The insertion of the tetrazole moiety ψ(CN 4) into the peptide sequence of [Leu 5]enkephalin considerably changes the coordination ability of the ligand. Potentiometric and spectroscopic results indicate that if the tetrazole moiety is in a suitable position in the peptide chain, i.e. if it follows the third residue, an unusual stable CuH −1L species involving 4N coordination is formed in the physiological pH region. The tetrazole ψ(CN 4) ring provides one of these nitrogens. The data indicate that Cu(II) ions are strongly trapped inside a bent peptide backbone. However, the coordination mode involving the tetrazole ring nitrogen does not prevent the hydrolysis process under strongly basic conditions.

Partitioning of kinetic energy to internal energy in the low energy collision-induced dissociations of proton-bound dimers of polypeptides

Collision-induced dissociation (CID) of the proton-bound dimers of a set of pentapeptides (leucine enkephalin analogs) generated by electrospray ionization is studied as a function of collision energy under conditions of single collisions with argon. As the collision energy is increased, the abundances of the two protonated peptides become more similar, indicating an increase in internal energy deposition. The effective temperature ( T eff) of the cluster ions is calculated by the kinetic method and found to increase approximately linearly with collision energy. Knowing the fragmentation thermochemistry, the ion internal energy is characterized using the kinetic method. The partitioning quotient for the conversion of laboratory kinetic energy into internal energy for these cluster ions is 2% to 5% in the 50 eV to 200 eV collision energy range. Average relative standard deviations of multiple measurements of partitioning quotients are around 15% and are mainly due to uncertainties in ion abundance ratios. Unimolecular dissociation Rice–Ramsperger–Kassel–Marcus (RRKM) theory is used to calculate the relationship between the fragment ion abundance ratio and the total internal energy of the cluster ions. Comparison of these data with experiment allows the energy partitioning behavior to be characterized independently and more accurately. The partitioning quotient obtained in this way ranges from 2 ± 1.0% (uncertainty is the standard derivation of multiple measurements) to 5 ± 1.0%. These data are consistent with either an impulsive collisional activation mechanism or with collision complex formation.

A New Efficient Method for S–CH2–S Bond Formation and Its Application to a Djenkolic Acid-Containing Cyclic Enkephalin Analog

Abstract An efficient methylene insertion reaction to construct an S–CH2–S bridge between two cysteine residues occurred when the thiol-protecting dimethylphosphinothioyl (Mpt) group of Z–Cys(Mpt)–OMe was removed with tetrabutylammonium fluoride hydrate in CH2Cl2. The thiol-free form gave similar results, albeit the yields were somewhat lower. In both cases, the best yields were obtained using 2 molar amounts of the reagent. Higher amounts of the reagent reduced the yield because of dehydroalanine formation. In the case of penicillamine, the thiol-free form was better in reactivity than the S-Mpt form, which required double the amount of the reagent to give the same yield. The reaction was successfully used in a synthesis of a cyclic enkephalin analog with the S–CH2–S bridge.

Cyclic enkephalin analogs that are hybrids of DPDPE-related peptides and metenkephalin-Arg-Gly-Leu: prohormone analogs that retain good potency and selectivity for delta opioid receptors.

We report here on the binding affinity and bioassay results of cyclic enkephalin analogs comprising a cyclic moiety and C-terminal fragment of MERGL, where ME denotes methionine enkephalin. MERGL (YGGFMRGL) has been suggested to be cleaved enzymatically by membrane-bound enkephalinase 24.11 to leave ME and the tripeptide RGL. In our study we have synthesized hybrids of DPDPE or DPLCE and the C-terminal tripeptide RGL in order to mimic a prohormone able to cross the blood-brain barrier. The study has shown that of the homologs presented here, analogs of DPLCE often are more potent at delta opioid receptors both in binding affinity and in bioactivity at the MVD, than DPDPE. Our hypothesis that hybrids (consisting of the drug and the spacer for the carrier) could be designed which would either have no opioid activity or, alternatively, be by themselves very active, has been verified.